Objective To establish the rational antibiotic treatment by accurate screening of high level aminoglycoside (HLA) resistant enterococci in clinical laboratory. Methods A single HLA genta micin 500 ?g/ml and streptomycin 200 ?g/ml for agar dilution method, and gentamicin 120 ?g/disk and streptomycin 300 ?g/disk for disk diffusion method were used as parallel screening tests to detect high level aminoglycoside resistance in 172 enterococci strains. Antibiotic time kill test was performed to verify the accuracy and reliability of both methods.Results The resistant rates of the two methods for HLA gentamicin were 60.0% and 59.3%,respectively. While for HLA streptomycin, both were 61.1%. In disk diffusion tests of 172 enterococci strains, the resistant rates for penicillin, ampicillin and vancomycin were 16.9%, 14.0%, and 1.7%, respectively. ? lactamase from 80 strains enterococci were all negative. Conclusion High concentration aminoglycoside disk diffusion method is a simple, reliable method for screening the HLA resistant enterococci in clinical laboratory. The results can provide a rational base for physicians to treat enterococcal infections.